.gamma.-Glutamyltranspeptidase (hereinafter abbreviated as .gamma.-GTP) is an enzyme widely distributed in tissues of organisms and involved in the metabolism of .gamma.-glutamylpeptides. In the area of clinical laboratory testing, the .gamma.-GTP activity is an important test that is widely and routinely employed in the diagnosis of heptatic diseases such as hepatocirrhosis, alcoholic hepatitis, hepatoma and obstructive jaundice and in disease state monitoring in patients suffering from such diseases.
In reagents generally used for the determination of .gamma.-GTP activity, a .gamma.-glutamylamide, which is a synthetic substrate, is used as the .gamma.-glutamyl donor and the amine liberated by the action of .gamma.-GTP is determined. As typical examples, reagents in which .gamma.-glutamyl-p-nitroanilide is used as the substrate (cf. Japanese Patent Application (OPI) No. 99198/80 (the term "OPI" as used herein means an "unexamined published Japanese patent application")), and reagent systems in which .gamma.-glutamyl-.alpha.-naphthylamide is used as the substrate (cf. Japanese Patent Application (OPI) No. 6392/75) are known. With the former substrate, either the p-nitroaniline liberated by the action of .gamma.-GTP is directly assayed by colorimetry or the p-nitroaniline is converted into a red-colored substance using an aldehyde such as p-dimethylaminocinnamaldehyde, which is then assayed by colorimetry. In the reagents using .gamma.-glutamyl .alpha.-naphthylamide as the substrate, the naphthylamine liberated by the action of .gamma.-GTP is converted to a diazonium salt, which is then assayed by colorimetry, or is oxidatively coupled with 3-methyl-2-benzothiazolinone hydrazone to give a reddish-purple substance, which is then assayed colorimetrically.
Reagents for .gamma.-GTP activity assay which use a conjugate enzyme have also been proposed. In one of them, a .gamma.-glutamyl-organic group-substituted methylamide compound is used as the substrate, and the substituted methylamine compound formed by the action of .gamma.-GTP is brought into contact with amine oxidase, and the consumption of dissolved oxygen or yield of hydrogen peroxide or ammonia resulting from the progress of the enzymatic reaction is quantitatively determined by a conventional method (cf. British Patent 2,103,607). In Japanese Patent Application (OPI) No. 34200/85, it is proposed that .gamma.-glutamyl-L-glutamic acid, which is close in nature to naturally occurring substances, be used as the substrate, that the L-glutamate liberated by the action of .gamma.-GTP be brought into contact with L-glutamate oxidase, and that the consumption of dissolved oxygen or yield of hydrogen peroxide, ammonia or .alpha.-ketoglutarate be determined by a conventional method.
However, each of the reagents for .gamma.-GTP assay using the above-mentioned synthetic substrates has its drawbacks. For instance, .gamma.-glutamyl-p-nitroanilide and similar substances have very poor solubility in the pH range in which these compounds are stable and in the neighborhood of the optimum pH for .gamma.-GTP reaction, making it difficult to use the substrates in sufficient amounts. Furthermore, these synthetic substrates are not very stable. In addition, they cause absorbance changes in the wavelength region in which bilirubin and/or hemolysis (hemoglobin) is significant and this makes it difficult to obtain exact assay results. The assay reagents using .gamma.-glutamyl-.alpha.-naphthylamide as a substrate have the further disadvantage that the procedure is complicated.
A number of measures for overcoming these drawbacks have been proposed (e.g., use of surfactants (cf. Japanese Patent Application (OPI) No. 103352/83), use of variously substituted substrates (cf. British Patent 2,103,607 and European Patent 152,274/85), addition of cyclodextrin (cf. U.S. Pat. No. 4,511,651). However, the problems have not yet been solved to an extent such that the measures can be widely practiced in routine clinical testing.
The enzymatic assay reagents which use amine oxidase or L-glutamate oxidase are also disadvantageous since these enzymes have relatively low substrate specificity, and the absorbance in the measuring wavelength region is strongly influenced by bilirubin and/or hemoglobin coexisting in blood or urine. They therefore lack reliability from the viewpoint of routine use in clinical laboratories.
Furthermore, each of the above-described conventional assay systems requires a complicated or troublesome procedure because of the necessity of using a standard substance with known activity.
Thus, it would be highly desirable to develop a reagent for the assay of .gamma.-GTP which uses, as the substrate, a peptide compound analogous to natural substrates for .gamma.-GTP permitting the .gamma.-GTP activity to be determined directly from the continuous course of reaction by a simple and easy procedure.